B cell lymphomas are neoplasms of mature lymphocytes which generally express immunoglobulin (Ig) on the cell surface. The Igs are clone specific and the variable regions contain determinants which may themselves be recognized as antigens or idiotopes. The idiotypic Ig therefore represents a unique tumor-specific antigen. (Wen Y J., et al. Eur. J. Immunol. 1997. 27: 1043-1047). Traditional treatments, such as chemotherapy and radiation therapy, can both induce remission in patients with low grade non-Hodgkin's lymphoma (NHL). However, despite this responsiveness to treatment, the majority of patients ultimately relapse and cannot be cured with these standard therapies and long-term outlook for this patients remains poor.
It is likely that the disease is essentially incurable, except may be by allogenic bone marrow transplantation. As the number of patients who would be suitable for such a therapeutic modality is extremely small, alternative methods of treatment are needed. An attractive approach would be to evoke the ability of the immune system to recognize and eliminate neoplastic cells while sparing normal cells. B-cell lymphomas represent ideal models for experimental tumor immunotherapy. In fact the Ig's idiotypes displayed on their cell surface represent attractive tumoral antigens since they are tumor-specific, belong to a well known family of molecules and it has been demonstrated that idiotype-specific immune response can be obtained also for self Igs. However, the efficacy of the anti-idiotypic vaccination is likely to be more effective in some histotypes than in others. Low grade B-cell lymphomas, as those with a small lymphocytic or follicular histotype, are expected to represent ideal targets because they i) generally show an indolent behaviour and are difficult to eradicate; ii) present a very low degree of clonal evolution; iii) express surface Igs at high levels; iiii) do not secrete surface immunoglobulins.
On the other hand, anti-idiotypic vaccination is expected to be less effective against lymphoblastoid and other high-grade lymphomas, which do not express immunoglobulins on the surface; moreover, these types are very aggressive and may show a high degree of clonal evolution (30-40%).
Although the traditional approach to immunization, i.e., sub-cutaneous immunization with the whole immunoglobulin by B-cell lymphoma mixed with adjuvant, was proven to be effective both in experimental models (George et al. J. Immunol., 138: 2168-2174, 1988), as well as in controlled clinical trials (Kwak et al. N. Engl. J. Med., 327: 1209-1215, 1992), such an approach was hampered by the need of the high amounts of purified protein that have to be prepared and certified for each case.
Several reports have indicated that the immunodominant epitopes of the clone specific Ig lies within the hypervariable regions and mainly within the third complementary determining region of immunoglobulin heavy chain (VH-CDR3) (Campbell et al. J. Immunol., 139: 2825-2833, 1987/Watanabe et al. J. Immunol., 151: 2871-2876, 1993). Furthermore, many observations suggest that immunization with whole protein may in some case produce an antibody response restricted to short linear epitopes, and consequently inadequate, rather than recognizing the three-dimensional structure of the protein itself (Kawaguchi et al. J. Biol. Chem., 264: 5762-5767, 1989). Conversely, immunization with short peptides (8-20 aa residues long) can result in production of antibodies that recognize the corresponding linear epitope on a protein without need for conjugation to carriers, provided that the short peptide behaves as a complete antigen (i.e. contains sequences able to bind Class II MHC molecules and to engage the T-cell receptor).
Indeed, it was demonstrated that a short peptide encompassing the VH-CDR3 region of a human lymphoma specific IgM was able to promote the in vitro proliferation of specific CD4+ and CD8+ cells, capable to lyse the autologous lymphoma cells (Wen et al. Eur. J. Immunol., 27: 1043-1047, 1997). The clinical relevance of these results obtained in vitro was confirmed by the specific immune response obtained in a patient following vaccination with the peptide (Wen et al. Br. J. Haematol., 103: 663-668, 1998).
The development of the method of vaccination by means of direct injection of naked DNA into the muscle or subcutaneously, combined with the ability of easily identifying and cloning individual tumor-specific idiotypes, has improved the chances of exploiting these tumor antigens. This approach has already proven effective in inducing immune responses to several antigens mainly of viral origin, and it has been applied to therapy of experimental murine lymphomas where the Ig characterizing the lymphoma has been used for immunotherapy of the parental tumor via DNA based vaccination (Hawkins et al. Blood, 83: 3279-3288, 1994/Fazio V. M. Res. Virol., 148: 101-108, 1997/Stevenson et al. Immunol. Rev., 145: 211-228, 1995). Such a method has proven effective in eliciting anti-idiotype specific immune responses when whole Ig, or its variable regions engineered to be expressed on non-self Ig (Syringelas et al. Nature Med., 2: 1038-1041, 1996), germ-line light chains (Watanabe et al. J. Immunol., 151: 2871-2876, 1993) or toxin fusion protein (King et al. Nature Med., 11: 1281-1286, 1998) were used as encoded antigen.
Nevertheless, if this technology were to be transferred to clinical practice, such a time consuming work would still be needed for each patient to render this approach too expensive to be carried out on a regular clinical basis.
An efficient and cheap DNA-based method for inducing an immune response against B-lymphoproliferative disorders would be desirable; in particular if we consider that purified double strand DNA itself is not immunogenic (Parker S E, Borellini F. et al. Hum. Gene Ther., 10 (5): 741-58, 1999). Moreover, if we consider that the calculate rate of mutation would be 3000 times less than spontaneous mutation rate for mammalian genomes, the level of integration, if should occur, would not be considered to pose a significant safety concern. (Martin T., Parker S E, et al. Hum. Gene Ther. 10 (5): 759-68).